Dysfunction of hemodialysis vascular access is the single most important contributor to the morbidity and mortality of patients on chronic hemodialysis. The outcome for even the most favored vascular access, the arteriovenous fistula (AVF), is dismal with up to 60% of AVFs never functioning, and increasing subsets of once functional AVFs eventually ceasing to do so. AVF failure largely reflects 3 processes: neointimal hyperplasia, impaired vasorelaxation and aberrant arterial remodeling, and thrombosis. This application seeks to continue the examination of the basis for AVF dysfunction and the exploration of relevant therapeutic strategies. In the completed cycle, we utilized peripheral, surgically-created rodent AVF models, demonstrating that these models recapitulate the essential features of functional human AVFs, including increased blood flow, and the critical features of failing human AVFs, including neointimal hyperplasia, thrombosis, and induction of vasculopathic genes. In these models, we demonstrate activation of proinflammatory transcription factors (NF-?B and AP-1), and the upregulation of maladaptive, vasculopathic genes (MCP-1) and adaptive, vasoprotective genes (eNOS and HO-1). Our proposed aims, resting and building on findings made in the concluded cycle, include the following. AIM I. Hypothesis: The NOS system determines adaptation and injury in the AVF. Examination. Using the rat AVF model, this aim will examine the role of specific NOS isoforms, and whether the NOS cofactor, BH4, and superoxide anion scavenging determine the phenotype of the AVF. These studies will be complemented by strategies employing mutant mice to examine the roles of specific NOS isoforms, GTP cyclohydrolase (the BH4-synthesizing enzyme), and endogenous NOS inhibitor, asymmetric dimethylarginine (ADMA). AIM II. Hypothesis: HO and its products protect against AVF failure. Examination: This aim will determine whether the premature AVF failure in HO-1-/- mice involves impaired arterial blood flow and vascular reactivity, increased NF-?B and AP-1 activation, and/or tissue factor-dependent thrombosis. This aim will determine the effects of HO products (carbon monoxide and bile pigments) on AVF pathobiology in HO-1+/+ mice, and whether these products can attenuate the premature failure of AVFs in HO-1-/- mice; the effect of HO induction in protecting the AVF will also be assessed. Finally, the potential protective effects in the AVF of HO-2, the constitutive HO isozyme, will be determined. AIM III. Hypothesis: Mediators upstream and downstream of MCP-1 contribute to AVF failure. Examination: This aim will examine the role of intermediates upstream of MCP-1 mRNA, namely, NF-?B and AP-1, and intermediates downstream of MCP-1 mRNA, specifically, MCP-1 protein and the MCP-1 receptor (CCR2). As our findings in the AVF suggest that MCP-1 may exert its adverse effects via RANTES (CCL5), the role of CCL5 in AVF failure will be examined using CCL5-/- mice and a CCL5 inhibitor. This application thus examines how 3 fundamentally important systems in vascular biology determine AVF success or failure, and may disclose therapeutic avenues for small molecules expected shortly from the pharmaceutical industry.